This proposal is designed to continue the long-standing project in the applicant's laboratory focused on gaining new understandings of mechanisms that regulate the function of cardiac and skeletal muscle ryanodine receptor(RyR)/calcium release channels. These channels are required for excitation-contraction (EC)coupling in cardiac and skeletal muscles. Accomplishments of the project in the past four years include: 1) the discovery of components of the RyR macromolecular signaling complex that play key roles in modulating channel function via phosphorylation/dephosphorylation; 2) elucidation of the role of leucine/isoleucine zipper motifs in targeting regulatory proteins to the RyR channel complexes; 3) demonstration that phosphorylation of RyR channels by protein kinase A (PKA) decreases the binding affinity for the stabilizing subunit of the channel known as FKBP. Our preliminary data suggest that the effect of PKA phosphorylation of RyR2 on FKBP12.6 binding is mediated by electrostatic repulsion due to negative charges on FKBP12.6 and the negative charges of the phosphate group added to RyR2. In addition, the applicant has identified distinct sites on RyR2 for PKA and CamKII phosphorylation using site directed mutagenesis in the full length RyR2. Moreover, novel components of the RyR macromolecular complex have been identified including the phosphodiesterase PDE4D3. Important new reagents have been developed for this project including a knock-in mouse that expresses the mutant RyR2-S2808Athat cannot be PKA phosphorylated. Hence, the focus for the next phase of this project will be on three new aims designed to elucidate the mechanism of regulation of RyR channels by PKA and CamKII phosphorylation, and the role of components of the RyR macromolecular complex including PDE4D3 and targeting proteins for kinases and phosphatases. The applicant has proposed that dysregulation of the RyR2 channel due to PKA hyperphosphorylation results in "leaky" channels due to decreased binding of FKBP12.6 to the channel, and that a drug JTV519 can fix this leak by enhancing the binding affinity of FKBP12.6 to the channel resulting in improved cardiac function in heart failure, and prevention of cardiac arrhythmias. Thus, the proposed studies are highly relevant to human diseases including heart failure and cardiac arrhythmias.